Methods of treating intestinal ischemia using heparin-binding epidermal growth factor

ABSTRACT

The present invention provides methods of treating pathologic conditions associated with intestinal ischemia. In the methods, patients at risk for or suffering from intestinal ischemia are treated with a heparin-binding epidermal growth factor product.

This application is a continuation of 09/181,974 filed Sep. 29, 1998,now U.S. Pat. No. 6,191,109 which claims the benefit of the filing dateof United States provisional application Ser. No. 60/063,858 filed Oct.31, 1997 now ABN.

FIELD OF THE INVENTION

The present invention generally relates to prevention and/or treatmentof ischemia-induced intestinal injury. More particularly the inventionrelates to prevention and/or treatment of intestinal injury usingheparin-binding epidermal growth factor (HB-EGF) products.

BACKGROUND OF THE INVENTION

Hemorrhagic disorders and ischemic states are the two major classes ofgastrointestinal circulatory disorders. A sudden reduction in the bloodsupply to a tissue is considered to be an ischemic event. Intestinalischemic events continue to play a major role in the morbidity andmortality of numerous patients. Ischemic injury to the small intestineresults in mucosal destruction, bacterial translocation, andperforation. Parks et al., Am. J. Physiol., 250: G749-753 (1986)attributes much of the injury associated with ischemic episodes to thereperfusion phenomena that begin when blood flow is restored.Immediately after an ischemic event, the intestinal epithelium undergoesdesquamation with destruction of the lamina propria. At a cellularlevel, ischemia leads to depletion of ATP and loss of cytoskeletalintegrity. With return of the blood supply (i.e., reperfusion), there iscontinued destruction of the villus structures. These injuries manifestthemselves in disease states such as necrotizing enterocolitis and canlead to overwhelming sepsis and multisystem organ failure. Recovery froman ischemic event depends on rapid proliferation and migration ofintestinal epithelial cells to regenerate damaged villi. Restitutionrequires the presence of multiple substances, including cytokines,hormones, and growth factors. Dignass and Podolsky, Gastroenterology,105: 1323-1332 (1993) reports that transforming growth factor-α (TGF-α),interleukin-1β (IL-1β), interferon-γ (IFN-γ), and epidermal growthfactor (EGF) have been shown to enhance restitution, possibly throughincreased production of transforming growth factor-β (TGF-β). Thesesubstances act to remodel the intestine after injury and to modulate theinflammatory response.

HB-EGF was originally identified in 1990 as a macrophage-secretedheparin binding growth factor. Like other members of the EGF family,HB-EGF exerts its biological effects by binding to the erb class of EGFreceptor (EGF-R) molecules. However, unlike most members of the EGPfamily including EGF, HB-EGF binds heparin with a high affinity. Heparinappears to potentiate binding of HB-EGF to the signal-transducing EGF-R,and may also modulate the biologic effects of the growth factor ontarget cells, including cellular migration and proliferation. HB-EGF ismitogenic for fibroblasts, smooth muscle cells and epithelial cells, butnot for endothelial cells. In addition, HB-EGF is produced by epithelialcells and acts as an autocrine growth factor for these cells. It is aheat-resistant, cationic protein, with a molecular weight ofapproximately 22,000 kDa that elutes from heparin-affinitychromatography columns with 1.0 M NaCl.

The cloning of a cDNA encoding human HB-EGF (or HB-EHM) is described inHigashiyama et al., Science, 251: 936-939 (1991) and in a correspondinginternational patent application published under the Patent CooperationTreaty as International Publication No. WO 92/06705 on Apr. 30, 1992.Both publications are hereby incorporated by reference herein. Thesequence of the protein coding portion of the cDNA is set out in SEQ IDNO: 1 herein, while the deduced amino acid sequence is set out in SEQ IDNO: 2. Mature HB-EGF is a secreted protein that is processed from atransmembrane precursor molecule (pro-HB-EGF) via extracellularcleavage. The predicted amino acid sequence of the full length HB-EGFprecursor represents a 208 amino acid protein. A span of hydrophobicresidues following the translation-initiating methionine is consistentwith a secretion signal sequence. Two threonine residues (Thr⁷⁵ and Thr⁸⁵ in the precursor protein) are sites for O-glycosylation. MatureHB-EGF consists of at least 86 amino acids (which span residues 63-148of the precursor molecule), and several microheterogeneous forms ofHB-EGF, differing by truncations of 10, 11, 14 and 19 amino acids at theN-terminus have been identified. HB-EGF contains a C-terminal EGF-likedomain (amino acid residues 30 to 86 of the mature protein) in which thesix cysteine residues characteristic of the EGF family members areconserved and which is probably involved in receptor binding. HB-EGF hasan N-terminal extension (amino acid residues 1 to 29 of the matureprotein) containing a highly hydrophilic stretch of amino acids to whichmuch of its ability to bind heparin is attributed. Besner et al., GrowthFactors, 7: 289-296 (1992), which is hereby incorporated by referenceherein, identifies residues 20 to 25 and 36 to 41 of the mature HB-EGFprotein as involved in binding cell surface heparin sulfate andindicates that such binding mediates interaction of HB-EGF with the EGFreceptor.

The EGF family comprises at least five polypeptides: EGF, HB-EGF, TGF-α,amphiregulin (AR), and betacellulin. For reviews of the family, seeBarnard et al., Gastroenterology, 108: 564-580 (1995) and Prigent andLemoine, Prog. Growth Factor Res., 4: 1-24 (1992). The amino acidsequence homology of HB-EGF to the EGF family members is 40 (compared toEGF) to 53% (compared to AR) between the first and sixth cysteineresidues in the EGF-like domains, but HB-EGF exhibits lower homologywhen the full length sequences are compared. Overall, HB-EGF mostclosely resembles AR in that the two polypeptides exhibit the highesthomology, appear to have a similar number of amino acids, and includethe N-terminal extension of highly hydrophilic amino acids upstream ofthe EGF-like domain.

Administration of EGF to prevent tissue damage after an ischemic eventin the brains of gerbils has been reported in U.S. Pat. No. 5,057,494issued Oct. 15, 1991 to Sheffield. The patent projects that EGF“analogs” having greater than 50% homology to EGF may also be useful inpreventing tissue damage and that treatment of damage in myocardialtissue, renal tissue, spleen tissue, intestinal tissue, and lung tissuewith EGF or EGF analogs may be indicated. However, the patent includesno experimental data supporting such projections.

The small intestine receives the majority of its blood supply from theSMA, but also has a rich collateral network such that only extensiveperturbations of blood flow lead to pathologic states. Villa et al.,Gastroenterology, 110(4 Suppl): A372 (1996) reports that in a rat modelof intestinal ischemia in which thirty minutes of ischemia are caused byocclusion of the superior mesenteric artery (SMA), pre-treatment of theintestines with EGF attenuated the increase in intestinal permeabilitycompared to that in untreated rats. The intestinal permeability increaseis an early event in intestinal tissue changes during ischemia. Multipleanimal models, like that described in Villa et al., supra have been usedto study the effects of ischemic injury to the small bowel. Since thesmall intestine has such a rich vascular supply, researchers have usedcomplete SMA occlusion to study ischemic injury of the bowel. Animalswho experience total SMA occlusion suffer from extreme fluid loss anduniformly die from hypovolemia and sepsis, making models of this typeuseless for evaluating the recovery from intestinal ischemia.Nevertheless, the sequence of morphologic and physiologic changes in theintestines resulting from ischemic injury has remained an area ofintense examination.

Miyazaki et al., Biochem Biophys Res Comm, 226: 542-546 (1996) discussesthe increased expression in a rat gastric mucosal cell fine of HB-EGFand AR resulting from oxidative stress. The authors speculate that thetwo growth factors may trigger the series of reparative events followingacute injury (apparently ulceration) of the gastrointestinal tract. Todate, there has been no published report of administration of HB-EGF invivo for any purpose, much less to test its ability to protect thegastrointestinal tract from injury from an ischemic event.

The prevention and treatment of ischemic damage in the clinical settingtherefore continues to be a challenge in medicine. There thus exists aneed in the art for models for testing the effects of potentialmodulators of ischemic events and for methods of preventing and/ortreating ischemic damage, particularly ischemic damage to theintestines.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides methods of treatingpathological conditions associated with intestinal ischemia byadministering an HB-EGF product to patients.

As used herein, “BB-EGF product” includes HB-EGF proteins comprisingabout amino acid 63 to about amino acid 148 of SEQ ID NO: 2; HB-EGPproteins comprising about amino acid 73 to about amino acid 148 of SEQID NO: 2; HB-EGF proteins comprising about amino acid 74 to about aminoacid 148 of SEQ ID NO: 2; HB-EGF proteins comprising about amino acid 77to about amino acid 148 of SEQ ID NO: 2; HB-EGF proteins comprisingabout amino acid 82 to about amino acid 148 of SEQ ID NO: 2; HB-EGFproteins comprising a continuous series of amino acids of SEQ ID NO: 2which exhibit less than 50% homology to EGF and which are efficacious inthe rat model specified below; fusion proteins comprising the foregoingHB-EGF proteins; and the foregoing HB-EGF proteins includingconservative amino acid substitutions. Conservative amino acidsubstitions are understood by those skilled in the art. The HB-EGFproducts may be isolated from natural sources known in the art [e.g.,the U-937 cell line (ATCC CRL 1593)], chemically synthesized, orproduced by recombinant techniques such as disclosed in WO92/06705,supra, the disclosure of which is hereby incorporated by reference. Inorder to obtain HB-EGF products of the invention, HB-EGF precursorproteins may be proteolytically processed in situ. The HB-EGF productsmay be post-translationally modified depending on the cell chosen as asource for the products.

The administration of HB-EGF products is preferably accomplished with apharmaceutical composition comprising an HB-EGF product and apharmaceutically acceptable carrier. The carrier may be in a widevariety of forms depending on the route of administration. The route ofadministration may be oral, rectal, parenteral, or through a nasogastrictube. Examples of parenteral routes of administration are intravenous,intraperitoneal, intramuscular, or subcutaneous injection. The presentlypreferred route of administration is the oral route as the presentinvention contemplates that the acid stability of HB-EGF is a uniquefactor as compared to, for example, EGF. The HB-EGF pharmaceuticalcomposition may also include other ingrediants to aid solubility, or forbuffering or preservation purposes. Pharmaceutical compositioncontaining HB-EGF products comprises HB-EGF at a concentration of about0.5 to 10 mg/ml and preferably at a concentration of 1 mg/ml in saline.Addition of other bioactive compounds [e.g., antibiotics, free radicalscavenging or conversion materials (e.g., vitamin E, beta-carotene, BHT,ascorbic acid, and superoxide dimutase), fibrolynic agents (e.g.,plasminogen activators), and slow-release polymers] to the HB-EGFcompounds or separate administration of the other bioactive compounds isalso contemplated.

As used herein, “pathological conditions associated with intestinalischemia” includes conditions which directly or indirectly causeintestinal ischemia (e.g., premature birth; birth asphyxia; congenitalheart disease; cardiac disease; polycythemia; hypoxia; exchangetransfusions; low-flow states; atherosclerosis, embolisms or arterialspasms; ischemia resulting from vessel occlusions in other segments ofthe bowel; ischemic colitis; and intestinal torsion such as occurs ininfants and particularly in animals) and conditions which are directlyor indirectly caused by intestinal ischemia (e.g., necrotizingenterocolitis, shock, sepsis, and intestinal angina). Thus, the presentinvention contemplates administration of HB-EGF products to patients inneed of such treatment including patients at risk for intestinalischemia, patients suffering from intestinal ischemia, and patientsrecovering from intestinal ischemia. The administration of HB-EGF topatients is contemplated in both the pediatric and adult populations.

More particularly, the invention contemplates a method of reducingnecrosis associated with intestinal ischemia comprising administering anHB-EGF product to a patient at risk for, suffering from, or recoveringfrom intestinal ischemia. Also contemplated is a method of protectingintestinal epithelial cells from hypoxia comprising exposing the cellsto an HB-EGF product. Administration of, or exposure to, HB-EGF productsreduces lactate dehyrogenase efflux from intestinal epithelial cells,maintains F-actin structure in intestinal epithelial cells, increasesATP levels in intestinal epithelial cells, and induces proliferation ofintestinal epithelial cells.

In view of the efficacy of HB-EGF in protecting intestinal tissue fromischemic events, it is contemplated that HB-EGF has a similar protectiveeffect on myocardial, renal, spleen, lung, and liver tissue.

In another aspect, the invention provides a novel animal model ofintestinal ischemia, designated herein a model of “segmental” intestinalischemia, that is useful for evaluating the efficacy of putativetherapeutics. Mammals, preferably rats, are subjected to reversiblearterial occlusion, wherein a first order branch of the SMA and terminalcollateral branches are occluded. Preferably, the first order branch ofthe SMA is selected from the group consisting of the middle ileum andthe distal ilieum. Also preferably, six to seven terminal collateralbranches are occluded. Reversible occlusion may be accomplished by meanssuch as a micro-vascular clip or sutures.

DETAILED DESCRIPTION OF THE INVENTION

Practice of the methods of the present invention is illustrated in thefollowing examples wherein Example 1 describes production of HB-EGF byrecombinant techniques and demonstrations of activity of the recombinantprotein in various assays including cytoprotection of intestinalepithelial cells from hypoxia in vitro; Example 2 discloses a segmentalmodel of intestinal ischemia in the rat; Example 3 describes theefficacy of HB-EGP in treating intestinal ischemia in the rat model; andExample 4 details treatment of human adults and infants withpathological conditions associated with intestinal ischemia with HB-EGF.

EXAMPLE 1

The effects of treatment of rat intestinal epithelial cells withrecombinant human HB-EGF were examined. Specifically, HB-EGF was testedfor the ability to induce proliferation of intestinal endothelial cellsand also for the ability to protect intestinal epithelial cells fromhypoxia. Experiments were also performed to examine the mechanism ofHB-EGF cytoprotection. The rat intestinal epithelial cells IEC-18 (ATCCCRL 1589) were used in the experiments.

A. Production of recombinant human HB-EGF (rHB-EGF)

The maltose-binding protein (MBP) fusion system (New England Biolabs,Beverly, Mass.) was used to produce recombinant human HB-EGF. HB-EGFcDNA corresponding to nucleotides 220 to 444 of SEQ ID NO: 1 (encodingamino acids 74-148 of the 208-amino acid HB-EGF precursor molecule) wascloned into plasmid pMAL-c2 at the Xmnl and HindIII sites. E. colistrain BL21 (F ompT r_(B) ⁻m₈ ⁻) (Novagen, Madison, Wis.) containingthis construct was grown at 37° C. in LB broth (Gibco/BRL, Gaithersburg,Md.) containing 2 g/L glucose (Gibco/BRL) and 100 μg/ml ampicillin(Sigma, St. Louis, Mo.) to an OD₆₀₀ of 0.2-0.3. To induce expression,IPTG (Promega, Madison, Wis.) was added to a final concentration of 0.3mM. After a 3 hour incubation at 37° C., cells were harvested bycentrifugation (4,000×g, 20 minutes) and the cell pellet was resuspendedin MBP buffer (10 mM Tris-Ci, 200 mM NaCl, 1 mM EDTA) containing 1 mMPMSF (Sigma) and frozen overnight at 20° C. Thawed cell sample was lysedwith a french press (14,000 psi) and the insoluble fraction was removedby centrifugation (9,000×g, 30 minutes). The supernatant was passed overan amylose resin column (New England Biolabs) and fusion protein waseluted in MBP buffer containing 10 mM maltose (Sigma). rHB-EGF wascleaved from MBP with Factor Xa (0.5%, w/w) (Boehringer Mannheim,Indianapolis, Ind.) at 23° C. for 16 hours. Cleaved products wereapplied to a TSK-heparin 5PW column (8×75 mm, TosoHaas, Philadelphia,Pa.) that was equilibrated with buffer (10 mM Tris-HCI pH 7.4, 0.2MNaCl). The column was washed with equilibration buffer and boundproteins were eluted with a 40 ml linear grdient of 0.2-2.0M NaCl in 10mM Tris-HCI pH 7.4 at 1 ml/min using an FPLC system (Pharmacia LKBBiotechnology, Piscataway, N.J.). One milliliter fractions are collectedand assayed in an EGF radioreceptor assay essentially as described inBesner et al. (1992), supra. Fractions showing peak displacement of¹²⁵I-EGF binding were pooled, adjusted to contain 5% acetonitrile and0.1% trifluoroacetic acid and subjected to reverse phase HPLC (RP-HPLC).RP-HPLC was performed in a Hitachi (San Hose, Calif.) HPLC system usinga Vydac C₄ column (0.46×25 cm, 5 μm particle size; The SeparationsGroup, Hesperia, Calif.) that was equilibrated with water containing 5%acetonitrile and 0.1% trifluoroacetic acid. The HB-EGF sample wasinjected onto the column and the column was eluted using a multilineargradient of 5% acetonitrile (isocratic) for 5 minutes, 5-15%acetonitrile over 5 minutes, 15-40% over 120 minutes, 40-90% over 1minute, 90% isocratic for 10 minutes, 90-5% over 1 minute, and 5%isocratic for 33 minutes. The flow rate was 1 ml/min throughout and 1 mlfractions were collected. After these purification steps, the absorbancepeak eluting at 19% acetonitrile is shown by SDS-PAGE to be a singleband migrating at approximately 13 kDA. NH₂-terminal amino acidsequencing of the pure protein produced in this fashion confirms thesequence for HB-EGF. The rHB-EGF was biologically active in theEGF-radioreceptor assay and a Balb/c 3T3 DNA synthesis assay[essentially as described in Besner et al., Cell Regulation, 1: 811-819(1990)].

B. Recombinant HB-EGF is mitogenic for intestinal epithelial cells

IEC-18 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)containing 5% fetal bovine serum (FBS), 50 μg/ml penicillin and 50units/ml streptomycin in a humidified atmosphere of 10% CO₂ at 37° C.Cells were then seeded at a density of 1×10₅ cells/well in a 24 wellplate (2 ml/well). After a 24 hour incubation, medium was changed toDMEM/1% FBS (2 ml/well) and cells were allowed to incubate for anadditional 24 hours. Cells in four of the wells were trypsinized andcounted at Day 0 using a Coulter Counter®. rHB-EGF was added toduplicate wells on Day 0 at a concentration of 100 ng/ml. Thisconcentration of rHB-EGF was determined as optimal from preliminarydose-response curves for rHB-EGF-stimulated proliferation of IEC-18cells. Cells in duplicate wells were trypsinized and counted on days 1through 5.

Under normoxic conditions, rHB-EGF-treated cells had 1.85 fold greaterincrease in number compared to non-treated cells by day 4 (p<0.05).Further, the mitogenic response of IEC-18 cells to rHB-EGF wasdose-dependent, with maximal stimulation by 100 ng/ml.

C. Recombinant HB-EGF to protects intestinal epithelial cells againsthypoxia

Lactate dehyrogenase (LDH) efflux was used as a measure of cell injuryafter hypoxia. IEC-18 cells were seeded at a density of 5×10⁴ cells/wellin DMEM/5% FBS in 24 well plates (2 ml/well). After 24 hours, medium waschanged to DMEM/1% FBS (2 ml/well). This plating density resulted inapproximately 75% confluency. After an additional 24 hour incubationperiod, the medium was replaced with Kreb's buffer (116 mM NaCl, 1.0 mMNaH₂PO₄, 25.0 mM NaHCO₃, 5.4 mM KCl, 1.8 mM CaCl₂, and 0.8 mM MgSO₄) andthe cells were placed in an anaerobic incubator with FiO₂≦1%. Aliquots(50 μl) of media were removed at specific time intervals and wereassayed for LDH efflux using a Cytox 96 assay. At the end of theexperiment, cells were lysed with PBS/0.1% Triton-100 and total LDHcontent was determined. LDH efflux was expressed as the percentage oftotal LDH activity. Based on preliminary experiments which demonstratedapproximately 20-25% cell death after 10 hours of hypoxia and 100% celldeath after 14 hours of hypoxia, a 10 hour anaerobic period was used inthe subsequent studies.

To test the growth factor effects, 100 ng/ml rHB-EGF was added to somewells 12 hours prior to the initiation of hypoxia. After 10 hours ofhypoxia, plates were removed from the anaerobic chamber, medium changedto DMEM/5% FBS, and cells were allowed to recover for 48 hours. Duringrecovery, some wells received additional (post-hypoxia) rHB-EGF (100ng/ml) treatment. Aliquots (50 μl) of media were removed from the wellsat 0, 12, 24, 36, and 48 hours of recovery and LDH efflux was measured.

Intestinal cells that received rHB-EGF either pre-hypoxia or both pre-and post-hypoxia showed a significantly lower LDH release duringrecovery from hypoxia compared to non-treated cells. Although there wasvery little LDH efflux immediately after hypoxia, by 48 hours,non-treated cells had an LDH release of 22.8% compared to 7.48% forcells that had been pre-treated with HB-EGF (p<0.009) or to 9.1% forcells that had been both pre-treated and post-treated with HB-EGF(P<0.009). Cells that received HB-EGF during only the post-hypoxicperiod did not have a significantly lower LDH release compared to cellsthat were not treated with HB-EGF.

D. Effects of rHB-EGF on cytoskeletal structure, ATP stores, andpost-hypoxia proliferation

To examine IEC-18 cytoskeletal structure, IEC-18 cells were seeded at5×10³ cells/well in DMEM/5% FBS (500 μl/chamber) in 8-well chamberslides. Cells were incubated for 24 hours after which the medium waschanged to DMEM/1% FBS. rHB-EGF (100 ng/ml) was then added to specificwells. After an additional 12 hours, medium was changed to Kreb's bufferand cells were placed in the anaerobic chamber for 10 hours. Followingthis, medium was changed to DMEM/5% FBS and cells were incubated for anadditional 24 or 48 hours. Medium was aspirated from the chambers, cellswere fixed for thirty minutes in 10% formalin (buffered with PBS), andslides were washed twice in PBS. Cells were simultaneously stained withrhodamine phalloidin to detect filamentous (F) actin and Dnase Ifluorescein to detect globular (G) actin. Confocal analysis of the cellswas performed with the Zeiss LSM inverted microscope using akrypton/argon mixed gas laser and a Zeiss filter set, utilizingemissions filters of 568 and 488. Images were digitally recorded.

Under normal circumstances, monomeric G-actin is polymerized to produceF-actin in an AT-dependent manner. F-actin staining of cells is presentin the cortical region (peripheral) in cells having an intactcytoskeleton, whereas G-actin accumulation is indicative of cytoskeletalinjury. Immediately after anaerobic exposure, the respective proportionsof F-actin and G-actin were similar between rHB-EGF-treated andnon-treated cells. However, after 24 hours of recovery, IEC-18 cellsthat received rHB-EGF prior to anaerobic exposure maintained thecortical F-actin cytoskeletal structure compared to non-treated cellswhich had increase levels of peri-nuclear G-actin staining. By 48 hours,rHB-EGF-treated cells still maintained their F-actin structure, whereasnon-treated cells contained predominately G-actin with very littleF-actin.

To examine ATP stores in IEC-18 cells, the cells were seeded at adensity of 1×10⁵ cells/well in DMEM/5% FBS in 6 well palates (1ml/well). Cells were incubated for 24 hours after which the medium waschanged to DMEM/1% FBS. rHB-EGF (100 ng/ml) was then added to specificwells. After an additional 12 hours, medium was changed to Kreb's bufferand cells were placed in the anaerobic chamber for 10 hours. Cells werelysed with PBS/0.1% Triton-100 at 0, 24 and 48 hours of recovery. ATPcontent was measured using an ATP determination kit (Molecular Probes,Eugene, Oreg.). This assay allows quantification of ATP through aluciferin/luciferase-ATP reaction. Bioluminescence was measured with aluminometer (Model LB9501). Initial experiments demonstrated a linearrelationship (r²=0.960) between ATP (pmoles) and luminescence. A Bio-RadD_(c) protein assay was used to determine total protein content in thesamples. Optical density (OD) was measured with a microplate reader(Model EL312). Preliminary standard curves demonstrated a linearrelationship (r²=0.988) between protein (mg/ml) and OD.

Both non-treated and rHB-EGF treated cells had ATP levels in the 11-12nmole/mg range under normoxic conditions. rHB-EGF-treated andnon-treated cells had similar decreases in ATP levels in the immediatepost-hypoxic period (45% drop vs 50% drop, respectively). However,during the later recovery periods (24 and 48 hours), the HB-EGF-treatedcells exhibited a rise in their ATP levels (6.1 nmole/mg), whereas thenon-treated cells continued to have decreased ATP content (4.5nmole/mg).

Post-hypoxia IEC-18 cell proliferation was assessed with a CytoQuantfluorescence assay (Molecular Probes, Eugene, Oreg.). Preliminaryexperiments demonstrated a linear relationship (r²=0.978) between cellnumber and fluorescence. IEC-18 cells were seeded at 5×10³ cells/well inDMEM/5% FBS in 96-well plates (200 μl/well). After 24 hours ofincubation, medium was changed to DMEM/1% FBS (200 μl/well). Some wellsreceived HB-EGF (100 ng/ml) and the cells were incubated for anadditional 12 hours. Medium was then changed to Kreb's buffer and theplate was placed in the anaerobic chamber for 10 hours. At 0, 12, 36,and 72 hours of recovery, media was removed and plates were frozen at−70° C. for thirty minutes. The Cytoquant fluoroprobe was added afterincubating plate at room temperature for five minutes. Fluorescence wasmeasured in a Cytofluor fluorescent plate reader (Millipore, Bedford,Mass.).

Cytofluorometric measurement of post-hypoxia cellular proliferation ofIEC-18 cells showed that cells treated with HB-EGF had a 1.23 foldincrease in a cell number compared to non-treated cells by 72 hours ofrecovery from hypoxia (p<0.05).

The experimental results described above relating to the mitogenic andcytoprotective effects of HB-EGF were orally disclosed at the ColumbusSurgical Society Presidential Symposium on Jan. 18, 1997 and at theAnnual West Virginia University Resident's Forum on Mar. 7, 1997. Theexperimental results described above relating to LDH release weredisclosed orally and in abstract form at the Childrens' HospitalResearch Foundation Research Forum in Columbus, Ohio on Jun. 5, 1997.

HB-EGF was also demonstrated to preserve cytoskeletal structure andincrease cellular ATP levels in renal tubular epithelial cells subjectto hypoxia. The same cells also released a lower level of LDH duringrecovery than untreated cells.

The results of the in vitro experiments indicate that in addition tobeing a mitogen for intestinal epithelial cells, HB-EGF is also acytoprotective growth factor for these cells during recovery fromhypoxia. The in vitro cytoprotective effects of rHB-EGF can beexplained, at least in part, by increased cellular ATP levels inrHB-EGF-treated cells with resultant preservation of cytoskeletalstructure. An additional beneficial effect of this growth factor is thatafter the ischemic event, HB-EGF-treated cells have a higherproliferative rate than non-treated cells. The cytoprotective effects ofHB-EGF may be enhanced by its ability to bind to heparan sulfateproteoglycans expressed on the surface of intestinal cells. See Carey etal., J. Cell Biology, 117(1): 191-201 (1992) for a discussion of heparansulfate proteoglycans.

EXAMPLE 2

While HB-EGF protected intestinal epithelial cells from hypoxia invitro, there was no model described in the literature that was usefulfor examining the effect of HB-EGF on recovery from intestinal ischemiain vio. To determine whether HB-EGF was efficacious in protecting theintestines from the deleterious effects of ischemia in vivo, a novelanimal model of segmental intestinal ischemia was developed. The modelprovides the opportunity to study ischemia-reperfusion injuries inlocalized segments of bowel without the morbidity and mortalityassociated with total SMA occlusion in prior animal models. By occludinga first order branch of the superior mesenteric artery (SMA) and byselectively ligating terminal collateral branches, reproduciblesegmental intestinal ischemia was achieved. Bowel damage ranged fromalterations in the villus structure to frank hemorrhagic necrosis of theintestinal wall.

The operative procedure was performed as follows. A total of eighteenrats (age 7 to 9 weeks, 200-265 g) were induced with an intraperitonealinjection of Ketamine-HCl (10 mg/kg) and Xylazine (3 mg/kg). Intravenousaccess and/or fluid resuscitation were not necessary during theprocedure. The abdomen was shaved and painted with betadine. The animalwas placed supine on a warming pad set at 40° C., and positioned underan operating microscope. A midline skin incision was made, the lineaalba was opened, and the peritoneal cavity was entered. The smallintestine, cecum and proximal ascending colon were delivered into theoperative field and the superior mesenteric vein was identified. The SMAwas located postero-lateral to the vein and was exposed by carefullydissecting apart the mesentery with 0.5 mm micro-surgical forceps.Exposure of this artery and the subsequent steps were performed using anoperating microscope. Once the SMA was exposed, the first order brancheswere evaluated for possible sites of occlusion. Since the mesentericarcades are longer in the ileal segments, the middle and distal ileumwere used as target segments for arterial occlusion. Once a segment wasidentified, a micro-vascular atraumatic clip (2.0 mm) was placed on thefirst order mesenteric branch feeding this segment. After the clip wasplaced, the terminal arterial and venous arcade branches, both proximaland distal to the occluded arcade, were ligated with 5.0 silk sutures.To create a 5 cm segment of ischemic bowel, six to seven terminalbranches need to be ligated. Arterial occlusion was maintained for 1hour. A 4×4 gauze pad was placed over the bowel during this time and wasfrequently moistened with warm saline. Prior to removing the micro-clip,0.1% Evan's blue solution (1.5 cc) was injected into the renal vein witha 28 gauge needle to confirm non-perfusion of the ischemic segment. Themicro-clip was then removed and Evan's blue, at the same concentration,was injected into the contralateral renal vein to establish return offlow to the ischemic segment. The silk ligatures were left on theterminal branches. The abdomen was closed in a standard fashion. Animalswere then placed in a warm incubator (40° C.) until awake (approximately20 minutes) and were then transferred to individual cages. They receivedwater, but not food, during the post-operative period. Animals wereeuthanized with CO₂ and segments of intestine were removed forhistologic analysis at 6 hours after surgery in 6 animals and at 48hours after surgery in 12 animals.

All eighteen animals survived the operation. Gross changes of the bowelduring the arterial occlusion were noted to occur in stages. After 10-15minutes of ischemia the serosa loses its sheen, and after 15-20 minutesthe bowel wall becomes edematous. After 25-35 minutes the bowel colorchanges from pink to white and later develops a more dusky appearance.The proximal and distal ends of the segment, where the terminal arteriesand veins were ligated, have a more bluish appearance, and the smallerveins become dilated. Peristalsis of the affected segment ceases withinthe first 25 minutes of ischemia. As ischemic time increases, the bowelwall becomes increasingly edematous. Confirmation of ischemia was shownwith Evan's blue dye injection, with the dye taken up by the normallyperfused bowel, but not by the ischemic segment. Upon termination ofarterial occlusion, uptake of the blue dye throughout the previouslyischemic bowel confirmed resumption of flow to this area.

After euthanization, the abdomen was re-explored and the segment ofischemic bowel, as well as portions of intestine both proximal anddistal to the hypo-perfused segment, were excised and fixed inHistochoice™ for 12 hours. The segments were then cross-sectioned at 1mm intervals, processed in a standard fashion, and embedded in paraffin.Sections were H&E stained and examined using a standard lightmicroscope. All six animals that were sacrificed at the 6 hour timepoint showed minor intestinal villus structural change. Instead of thenormal elongated distal tip, the villi had more flattened tips withreduction of cytoplasmic content and absence of the brush border. Oftwelve animals sacrificed after 48 hours, all showed evidence of villustip necrosis, and five also developed areas of hemorrhagic transmuralnecrosis with extensive polymorphonuclear leukocyte infiltration.

Animal care and experimentation described above conformed to standardslisted in the National Institute of Health's Guide for the Care and Useof laboratory Animals. The experimental protocol was evaluated andapproved by the Institutional Animal Care and Use Committee ofChildren's Hospital (protocol # 01496AR). Each procedure was performedby a single operator without the need for additional assistance. Allprocedures were performed using sterile technique. The duration of theprocedure was 1¼-1½ hours, depending on the difficulty of thedissection.

In comparison to ischemia-reperfusion injury models involving total SMAocclusion the mortality rate of which approaches 100%, the segmentalmodel described herein had a 0% postoperative mortality in the first 48hours. The development of the segmental model thus allowed theinvestigation of the effects of potential therapeutic agents during therecovery period.

EXAMPLE 3

The effect of HB-EGF on intestinal ischemia in vivo was then examinedusing the segmental model.

A total of twelve rHB-EGF-treated and twelve control male Sprague-Dawleyrats (220-310 g) were studied. Segmental intestinal ischemia involving a4.5-5.0 cm length of distal ileum was produced as described in Example2. Arterial occlusion was maintained for 1 hour. Fifteen minutes priorto removing the microvascular clip, Evan's blue solution was injectedintravenously to confirm segmental intestinal S ischemia. Experimentalanimals then received HB-EGF (20 μg/ml) which was injectedintraluminally into the duodenum in a volume of 5 ml of phosphatebuffered saline (PBS), which led to filling of the entiregastrointestinal tract from the duodenum to the cecum. Control animalsreceived an injection of 5 ml of PBS only. At the end of the 1 hourischemic interval, the microvascular clamp was removed and the Evan'sblue solution was reinjected intravenously to confirm segmentalintestinal reperfusion. Animals received water but no foodpostoperatively. After 48 hours animals were sacrificed and necropsyperformed. The ischemic intestinal segment as well as the bowel justproximal and distal to the ischemic segment were excised. Tissues wereplaced in Histochoice fixative for 12 hours, cross-sectioned at randomintervals and embedded in paraffin. Sections were H & E stained andexamined using a standard light microscope. For each animal in theexperiment, 4-10 random sections of the ischemic bowel segment wereexamined for histologic injury. Results are presented in Table 1 below.

TABLE 1 # # sections Transmural Total animals examined Tip necrosisnecrosis injury Non- 12 92 51/92 (55%) 21/92 (23%) 72/92 treated (78%)rHB- 12 94 12/94 (13%) 0/94 (0%) 12/94 EGF- (13%) treated P < 0.05

Of the non-treated animals, 55% of the representative sections studiedhad villous tip necrosis, and 23% of the sections had transmuralnecrosis. In contrast, in the HB-EGF-treated animals, only 13% of thesections studied had villous tip necrosis, and none of the sectionsdisplayed transmural necrosis. Thus, intraluminal HB-EGF administration,in this case delivered 45 minutes after the initiation of the ischemicevent, protects the intestine from ischemic injury.

EXAMPLE 4

The following section exemplifies administration of HB-EGF to pediatricpatients and adult patients. Administration of HB-EGF is indicated inpatients at risk for or suffering from any pathological conditionassociated with ischemic injury.

A. Administration to pediatric patients

Intestinal injury related to an ischemic event is a major risk factorfor neonatal development of necrotizing enterocolitis (NEC). NECaccounts for approximately 15% of all deaths occurring after one week oflife in small premature infants. Although most babies who develop NECare born prematurely, approximately 10% of babies with NEC are full-terminfants. Babies with NEC often suffer severe consequences of the diseaseranging from loss of a portion of the intestinal tract to the entireintestinal tract. At present, there are no known therapies to decreasethe incidence of NEC in neonates.

Babies considered to be at risk for NEC are those who are premature(less than 36 weeks gestation) or those who are full-term but exhibit,e.g., prenatal asphyxia, shock, sepsis, or congenital heart disease. Thepresence and severity of NEC is graded using the staging system of Bellet al., J. Ped. Surg., 15:569 (1980) as follows:

Stage I Any one or more historical factors producing perinatal stress(Suspected Systemic manifestations - temperature instability, lethargy,NEC) apnea, bradycardia Gastrointestinal manifestations -poor feeding,increased pregavage residuals, emesis (may be bilious or test positivefor occult blood), mild abdominal distention, occult blood in stool (nofissure) Stage II Any one or more historical factors (Definite Abovesigns and symptoms plus persistant occult or gross NEC) gastrointestinalbleeding, marked abdominal distention Abdominal radiographs showingsignificant intestinal disten- tion with ileus, small-bowel separation(edema in bowel wall or peritoneal fluid), unchanging or persistent“rigid” bowel loops, pneumatosis intestinalls, portal venous gas StageIII Any one or more historical factors (Advanced Above sings andsymptoms plus deterioration of vital signs, NEC) evidence of septicshock, or marked gastrointestinal hemorrhage Abdominal radiographsshowing pneumoperitoneum in addition to findings listed for Stage II

Babies at risk for or exhibiting HB-EGF are treated as follows. Patientsreceive a daily liquid suspension of HB-EGF (1 mg/kg in saline). Themedications are delivered via a nasogastric tube if one is in place, ororally if there is no nasogastric tube in place.

B. Administration to adult patients

Adults also receive a daily liquid suspension containing HB-EGF (1mg/kg) to drink or through a nasogastric tube if necessary.

Numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe foregoing description. Consequently, the only limitations whichshould be placed on the invention are those which appear in thefollowing claims.

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We claim:
 1. A method of treating intestinal cell necrosis associatedwith intestinal ischemia in a patient in need thereof comprisingadministering to said patients an effective amount of heparin-bindingepidermal growth factor product, effective to reduce intestinal cellnecrosis.